Mutagenesis studies of the major benzo[a]pyrene N2-dG adduct in a 5'-TG versus a 5'-UG sequence: removal of the methyl group causes a modest decrease in the [G->T/G->A] mutational ratio

The potent mutagen/carcinogen benzo[a]pyrene (B[a]P) is metabolically activated to (+)-anti-B[a]PDE, which induces a full spectrum of mutations primarily at the G:C base pairs (e.g. GC-->TA, GC-->AT, etc.). Each of these mutations can be induced by its major adduct [+ta]-B[a]P-N(2)-dG, where DNA sequence context appears to influence both the quantitative and qualitative pattern of mutagenesis. We noted previously that 5'-TG sequences tend to have a higher fraction of G-->T mutations for both [+ta]-B[a]P-N(2)-dG and (+)-anti-B[a]PDE in comparison with 5'-CG, 5'-GG or 5'-AG sequences. To investigate a possible structural element for this trend, the role (if any) of the methyl group on the 5'-T is considered. Using adduct site-specific means, the [G-->T/G-->A] mutational ratio for [+ta]-B[a]P-N(2)-dG is determined to be approximately 1.08 in a 5'-TGT sequence, and approximately 0.60 in a 5'-UGT sequence. (G-->C mutations are minor.) Although this modest approximately 1.8-fold decrease in [G-->T/G-->A] ratio is statistically significant (P = 0.03), it suggests that the methyl group on the 5'-T is not the main reason why a 5'-T tends to enhance G-->T mutations. This study was prompted by an adduct conformational hypothesis, which predicted that the removal of the methyl group in a 5'-TG sequence would lower the fraction of G-->T mutations; however, the approximately 1.8-fold decrease is too small to do additional experiments to assess whether this conformational hypothesis, or other hypotheses, are the true cause of the decrease, which is discussed in this paper.     

Mutagenic activity of the 4,5- and 9,10-dihydrodiols of benzo[J]fluoranthene and their syn- and anti-dihydrodiol epoxides in salmonella typhimurium

The objective of this study was to determine the relative mutagenic activities of the major dihydrodiol metabolites of benzo[j]fluoranthene (B[j]F) and their corresponding syn- and anti-dihydrodiol epoxides. Salmonella typhimurium tester strains TA97a, TA98, and TA100 were used to evaluate the mutagenic potencies of the parent hydrocarbon and these suspect proximate and ultimate mutagenic metabolites. B[j]F and the trans-dihydrodiol metabolites were active only in the presence of an external metabolic activation system (S9) with the exception of the B[j]F-4,5-diol, which was weakly active in TA98 and TA100 in the absence of S9. The B[j]F-4,5-diol was more mutagenic than the B[j]F-9,10-diol in tester strains TA98 and TA100, whereas the opposite effect was observed in TA97a. In the absence of S9, the anti-B[j]F-4,5-diol epoxide was more mutagenic than the syn-B[j]F-4,5-diol epoxide and the syn- and anti-B[j]F-9,10-diol epoxides in tester strains TA97a and TA100. The exceptional mutagenic potency of the anti-B[j]F-4,5-diol epoxide in TA100 resembles that observed by epoxides located within a fjord, or by the anti-diol epoxides of bay region methylated polycyclic aromatic hydrocarbons. In contrast, the mutagenicity of the pseudo bay region dihydrodiol epoxides arising from the B[j]F-9,10-diol more closely resembles that observed with the classical bay region dihydrodiol epoxides of chrysene. In summary, both dihydrodiol metabolites of B[j]F are mutagenic in S. typhimurium, and the relative potency varies among the tester strains. The highest mutagenic response was achieved in tester strain TA100, which detects base-pair substitutions. The most potent direct-acting dihydrodiol epoxide in this tester strain was the anti-B[j]F-4,5-diol epoxide, which agrees with the results of mouse skin painting studies that indicate that the B[j]F-4,5-diol is more tumorigenic that the parent hydrocarbon or the B[j]F-9,10-diol. A cova-lent DNA adduct formed between the anti-B[j]F-4,5-diol epoxide and deoxyguanosine was the major species of DNA adduct formed in S. typhimurium. This adduct corresponds to the major DNA adduct formed in mouse skin following application B[j]F.     

Mutagenicity of a single 1-nitropyrene-DNA adduct N-(deoxyguanosin-8-yl)-1-aminopyrene in Escherichia coli located in a GGC sequence.

1-Nitropyrene, a common environmental pollutant, forms a major DNA adduct, N-(deoxyguanosin-8-yl)-1-amino- pyrene (dG(AP)). Mutational spectra of randomly introduced dG(AP) in Escherichia coli included different types of mutations, which depended on the base sequence surrounding the adduct. In earlier works we investigated the DNA sequence context effects of the adduct in repetitive CpG and non-repetitive CpGpC sequences. In the current work this adduct was incorporated into a non-repetitive GpGpC sequence in single-stranded M13mp7L2 DNA with the adduct located at either the 5' or 3' G. Potent genotoxicity of dG(AP) was evident from a significant reduction in the population of progeny phage following replication of these constructs in repair-competent E.coli cells. However, progeny derived from the 3'-G(AP) construct were much larger than those from the 5'-G(AP) construct. We suspect that a more facile translesion synthesis past the adduct at the 3' G relative to that at the 5' G, presumably due to a difference in conformation of dG(AP) in these two sites, might be responsible for this effect. With both adducted constructs, >95% of the progeny did not show any mutations at or near the adduct site, indicating highly efficient error-free translesion synthesis. However, a small population of mutants with one base deletions and base substitutions were detected. While the adduct induced -1 frameshifts (<1%) in each G site, base substitutions (1-2%), exhibiting predominantly G-->C transversions, were detected only when the adduct was located at the 5' G. A comparison of the data from this study with a prior study in the CpGpC sequence suggests that dG(AP) mutagenesis is highly sensitive to the local DNA sequence and that a 5'-pyrimidine base might be important for targeted base substitutions by this adduct in E.coli.     

Carcinogen-DNA adducts in human breast epithelial cells

Diet and environmental exposures are often regarded as significant etiologic factors in human breast cancer. Chemicals that may be involved in these exposures include heterocyclic amines, aromatic amines, and polycyclic aromatic hydrocarbons, which also serve as strong mammary carcinogens in different animal models. In this study, we chose to quantify the major DNA adducts derived from one member of each of these classes of carcinogens, that is, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 4-aminobiphenyl (ABP), and benzo[a]pyrene (B[a]P), respectively, in DNA isolated from exfoliated ductal epithelial cells in human breast milk. Milk was collected from healthy, nonsmoking mothers. The isolated DNA was digested to 3' nucleotides and subjected to (32)P-postlabeling. Adduct enrichment was achieved using Oasis Sep-Paks and the analyses were conducted by HPLC using radiometric detection. Critical to the analyses were the syntheses of bis(phosphate) standards for the C8-dG adducts of PhIP and ABP, and the N(2)-dG adduct of B[a]P, which were added to each reaction as UV markers. Of the 64 samples analyzed, adducts were found in 31 samples. Thirty samples contained detectable levels of PhIP adducts, with a mean value of 4.7 adducts/10(7) nucleotides; 18 were positive for ABP adducts with a mean value of 4.7 adducts/10(7) nucleotides; and 13 were found to contain B[a]P adducts with a mean level of 1.9 adducts/10(7) nucleotides. These data indicate that women are exposed to several classes of dietary and environmental carcinogens and that these carcinogens react with DNA in breast ductal epithelial cells, the cells from which most breast cancers arise.     

Pentachlorophenol potentiates benzo[a]pyrene DNA adduct formation in adult but not infant B6C3F1 male mice

The objective of this study is to determine whether pentachlorophenol (PCP) alters benzo[a]pyrene (B[a]P)-induced DNA adduct formation in infant and adult B6C3F1 male mice. Mice were exposed intraperitoneally to 55 microg B[a]P/g body weight (BW) alone and in combination with several doses of PCP in DMSO. The 32P-postlabeling assay was used to analyze for (+/-) anti-7,8-diol-9,10-epoxide-B[a]P-N(2)deoxyguanosine (BPDE-N(2)G) adducts formed in liver and lung DNA. Hepatic DNA also was analyzed for 8-hydroxy-2'-deoxyguanosine (8-OHdG) base damage in mice exposed to PCP. 8-OHdG was not detected at any dose of PCP in infant or adult mice. PCP exhibited an antagonistic effect on BPDE-N(2)G accumulation in infant mice exposed to B[a]P in combination with 50 microg PCP/g BW at both 12 and 24 hr. Comparatively, BPDE-N(2)G adducts were increased in adult mice exposed to binary mixtures at 24 hr in both hepatic and lung DNA (P < 0.05). Multiple comparison analysis between infant and adult mice revealed that adduct levels in infants exposed to B[a]P alone or in combination with PCP were not different from those observed in adult mice exposed to B[a]P. However, a significant increase in adducts was observed in adult mice exposed to a combination of B[a]P and PCP compared to that in all other treatment groups (P < 0.05). These results suggest that PCP alters the metabolism of B[a]P in both infant and adult mice through different mechanisms, and that infants are not susceptible to the potentiating effects of PCP observed in adult mice.     

Influence of adduct position and sequence length on the ligation of oligonucleotides containing benzo[c]phenanthrene diol epoxide-deoxyadenosine adducts into M13mp7L2

The adduct that would arise from cis opening of (+)-(1S,2R,3R, 4S)-3,4-dihydroxy-1,2-epoxy-benzo[c]phenan-threne (benzo[c]phenanthrene diol epoxide-2, where the benzylic hydroxyl group and the epoxide oxygen are trans) by the exocyclic N6-amino group of deoxyadenosine was incorporated at the marked site into four oligonucleotides, 5'-CAGA*TTTAGAGTCTGC-3', 5'-CAGTGCAGA*TTTAGAG-3', 5'-GTGCAGA*TTTAGA-3' and 5'-TGCAGA*TTTA-3'. The oligonucleotides were inserted into M13mp7L2 and the vector transfected into SOS-induced Escherichia coli SMH77 which were then plated on agar plates. The experiments reported here were designed to test the effect of the lesion position (the marked A in the sequences above) on the ligation efficiency of the insert and the frequency of failed constructs, as well as any possible effects on the mutagenic consequences of the lesion. The construct survival was estimated from the number of plaques formed following transformation, and mutation frequencies were estimated from sequencing of randomly picked plaques. Moving the adduct site to the middle of the sequence increased considerably the ligation efficiency regardless of the length of the inserted oligonucleotide, and changing the insert length or the adduct location did not markedly affect the frequency (40-58.6%) or distribution of mutations observed. Thus, so long as the local sequence (five or six bases surrounding the adduct) remains constant, the size of the oligonucleotide insert and the position of the adduct in it can be adjusted to give optimal ligation efficiency without altering the mutagenic consequences of the lesion.     

Stereochemical effects in an insect repellent.

Racemic 1-[3-cyclohexen-1-ylcarbonyl]-2-methylpiperidine repels blood-feeding arthropods such as mosquitoes, chiggers, and ticks. The compound contains two asymmetric carbon atoms and the racemate consists of four stereoisomers. Quantitative mosquito bioassays using Aedes aegypti (L.) showed that (1S,2'S) and (1R,2'S) configurations were 2.8-3.1 and 1.6-1.8 times more effective, respectively, than the other two stereoisomers in reducing mosquito bites. (1S,2'S) was 2.5 more repellent than the racemate. Biological data show that an interaction of the (2'S)-2-methylpiperidine configuration with a repellent receptor system in A. aegypti is apparently important to repellent activity. Nuclear magnetic resonance spectra and molecular mechanics calculations for the stereoisomers provided insight into the conformation of the (2'S)-group. Results indicate that enhanced repellent effects can be realized through formulation of the most active stereoisomers of the compound.     

Characterization of a major aromatic DNA adduct detected in human breast tissues

A bulky DNA adduct (Spot 1) was previously detected in normal adjacent breast tissues of 41% (36/87) of women with breast cancer and in none (0/29) of the noncancer controls by (32)P-postlabeling. To characterize this adduct, it was chromatographically compared with DNA adduct profiles generated in several in vitro and in vivo experimental systems. First, MCF-7 cells were exposed to a number of chemical carcinogens, that is, benzo[a]pyrene (B[a]P), 4-OH-B[a]P, 9-OH-B[a]P, 11-OH-B[a]P, B[a]P-trans-4,5-dihydrodiol, 1-nitropyrene, 6-nitrochrysene, dibenzo[a,l]pyrene, benzo[c]phenanthrene, dibenzo[a,h]anthracene, 3-methylcholanthrene, and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. Spot 1 was detected as a minor adduct in cells treated with B[a]P but not other compounds. Second, to determine whether Spot 1 is derived from lipid peroxidation products or estrogen metabolites, it was compared with adduct profiles of cells or DNAs exposed to 17beta-estradiol, 4-hydroxy estradiol, 4-hydroxynonenal, or oxidized oat oil. Spot 1 was not detectable in these samples. In addition, Spot 1 did not comigrate with the 1,N(2)-ethenodeoxyguanosine adduct standard. Third, to explore the mechanism of Spot 1 formation, it was compared with adduct profiles detected in DNA or mononucleotides reacted with BPDE, 1-OH-7,8-dihydrodiol of B[a]P, and 3-OH-7,8-dihydrodiol of B[a]P as well as in rats orally treated with B[a]P. Spot 1 comigrated with a minor adduct in BPDE-treated DNA during anion exchange rechromatography but these two adducts were separated by partition chromatography. Spot 1 also behaved in a manner that was very similar to that of the polar B[a]P adducts detected in rat liver, but the two adducts were separated by HPLC. Fourth, Spot 1 was compared with CD1 mice exposed to 7H-benzo[c]fluorene (B[c]F). Spot 1 from some patients comigrated with a major adduct induced by B[c]F. Finally, we found that the presence of Spot 1 in human breast tissues was not related to smoking status but, rather, with CYP1A1 MspI polymorphism. The CYP1A1 mutant carriers had a significantly higher frequency of this adduct than did the wild-type genotypes. Furthermore, individuals with Spot 1 had a significantly higher staining intensity for BPDE-PAH adducts in their tissue sections than those without it. These results demonstrate that this major bulky DNA adduct detected in human breast tissues is related to PAH exposure.     

Antibacterial activity and mutagenicity studies of water-soluble phosphazene high polymers.

Eight water-soluble phosphazene high polymers, [NPR2]n (R, organic, water-solubilizing side-group; n, approx: 15,000) and the small-molecule counterparts of the polymers were examined for antibacterial activity against six different strains of bacteria (Escherichia coli, Salmonella typhimurium (TA 100), Salmonella pullorum, Streptococcus faecalis, Bacillus subtilis and Pseudomonas aeruginosa). Antibacterial testing was carried out by measuring zones of inhibition and changes in solution turbidity over time. In addition, the antibacterial activity of the surfaces of cross-linked poly[di(methoxyethoxyethoxy)phosphazene] (MEEP) hydrogels were investigated. A number of the high polymers, as well as the MEEP hydrogels, impeded bacterial growth. Only E. coli was unaffected by the phosphazenes. A possible explanation for the antibacterial character of the polymers is presented. The same compounds were monitored for potential mutagenic activity using the Salmonella typhimurium tester strains TA 100 and TA 98. None of the high polymers or their small-molecule analogues showed mutagenic activity in either strain of Salmonella at the concentrations tested. The use of these materials as coatings for artificial implants is discussed.     

Effect of the co-carcinogen benzo[e] pyrene on microsome-mediated chemical mutagenesis in salmonella typhimurium

Chemical agents that possess the ability to alter tumorigenicity of carcinogens (administered at subthreshold dose) constitute a major health hazard. We have employed the Ames Salmonella assay to examine the effect of co-carcinogenic benzo[e] pyrene (B[e]P) on microsome-mediated chemical mutagenesis. B[e]P enhanced the mutagenic activity induced by 2-acetylaminofluorene (2-AAF) and benzo[a] pyrene (B[a]P) in strains TA1538 and TA98. Enhancement was also noted with N-hydroxy-2-AAF (the proximal metabolite of 2-AAF) but not with an ultimate carcinogenic form (N-acetoxy-2-AAF). These results suggest the use of this approach to detect chemical agents that possess the ability to alter the activity of mutagenic or carcinogenic chemicals.     

Studies of dose distribution, premutagenic events and mutation frequencies for benzo[a]pyrene aiming at low dose cancer risk estimation.

Cancer risk assessment of polycyclic aromatic hydrocarbons (PAH) is complicated by several of these compounds exerting a promoter action leading to high tumour incidences at high doses. Cancer risks at low doses corresponding to the uptake from air and food in the general environment would best be estimated on the basis of measurement of in vivo target doses of genotoxic (mutagenic) intermediates and a determination of mutation frequency per unit of dose. In experiments ultimately aiming at a risk assessment of environmental PAH from in vivo doses benzo[a]pyrene (BaP) was chosen as a model. gamma-Radiation has earlier been used as a reference standard in cancer risk estimation of genotoxic chemicals where dose equivalents (rad-equivalents) have been shown to give reliable risk estimates for several alkylating agents. Variation in dose of BaP diolepoxide between organs was studied by measurement of deoxyguanosine-N(2) adducts in DNA after administration of BaP by gavage to mice of a strain with reduced DNA repair (Xpa(-/-)). The adduct levels in spleen, forestomach, stomach and small intestine were approximately the same; with the adduct level in spleen as reference it was twice as high in liver and lung and about half as high in colon tissue. A chemical or radiation dose is proportional to the cumulative frequency of putatively premutagenic changes (premutagenic hits) in DNA. The mutation frequency per premutagenic hit (genotoxic chemicals) and per unit of dose (gamma-radiation) were calculated from acutely exposed V79 cells in order to determine the mutagenic effectiveness of each agent. Based on the mutagenic effectiveness determined in this study 10(-4) Gy can be regarded equally effective in causing phenotypically expressed HPRT mutations as the dose of BaP which causes the formation of one deoxyguanosine-N(2) adduct per cell.     

Effect of the co-carcinogen benzo[e]pyrene on microsome-mediated chemical mutagenesis in Salmonella typhimurium.

Chemical agents that possess the ability to alter tumorigenicity of carcinogens (administered at subthreshold dose) constitute a major health hazard. We have employed the Ames Salmonella assay to examine the effect of co-carcinogenic benzo[e]pyrene (B[e]P) on microsome-mediated chemical mutagenesis. B[e]P enhanced the mutagenic activity induced by 2-acetylaminofluorene (2-AAF) and benzo[a]pyrene (B[a]P) in strains TA1538 and TA98. Enhancement was also noted with N-hydroxy-2-AAF (the proximal metabolite of 2-AAF) but not with an ultimate carcinogenic form (N-acetoxy-2-AAF). These results suggest the use of this approach to detect chemical agents that possess the ability to alter the activity of mutagenic or carcinogenic chemicals.     

Influences of DNA isolation and RNA contamination on carcinogen-DNA adduct analysis by 32P-postlabeling

³²P-Postlabeling is a widely applied assay for the analysis of carcinogen-DNA adducts. Optimization of most steps in this assay has been given attention, but influences of DNA isolation and DNA purity on adduct quantitation have not been investigated systematically. In this study, DNA was isolated from human lymphocytes exposed to benzo[a]pyrene (B[a]P, 10 μM) for 18 hr and from liver of rats i.p.-treated with B[a]P (10 mg/kg body weight) using two different DNA isolation methods: a phenol-extraction and a salting-out procedure. Subsequently, DNA was analysed by nuclease P1 (NP1) or butanol-enriched ³²P-postlabeling. Influences of RNA contamination were studied by labeling RNA isolated from in vitro exposed lymphocytes. In the in vitro experiment, DNA adduct levels were significantly higher using the salting-out procedure (63.2 ± 13.7 adducts per 10⁸ nucleotides, n = 9) as compared with the phenol-extraction (14.3 ± 0.8). RNA was ∼4 times less efficiently labeled as compared to DNA. Nonetheless, RNA contamination of DNA samples may result in an overestimation of DNA adduct levels when butanol enrichment is used, because RNA adduct levels seemed to be substantially higher than DNA adduct levels in the same cells. DNA adduct analysis by nuclease P1 enrichment is probably less affected, since RNA adducts appeared to be NP1 sensitive. In vivo, three different adducts were found by NP1 enriched ³²P-postlabeling in the liver of B[a]P-exposed rats. Again, DNA adduct levels were significantly higher using salting out as compared to phenol extraction for the adduct which comigrated with the BPDE-DNA adduct standard (adduct 1) and an unknown adduct (adduct 2). However, the results were the opposite for another B[a]P-derived DNA adduct (adduct 3). Our results suggest that differences in DNA isolation procedures as well as RNA contamination influence quantitative DNA adduct analysis by ³²P-postlabeling. Environ. Mol. Mutagen. 32: 344–350, 1998 © 1998 Wiley-Liss, Inc.     

Time- and dose-dependent DNA binding of PAHs derived from diesel particle extracts, benzo[a]pyrene and 5-methychrysene in a human mammary carcinoma cell line (MCF-7).

Cultures of a human mammary carcinoma cell line (MCF-7) were exposed to the soluble organic fraction of diesel particle emissions, benzo[a]pyrene (B[a]P) and 5-methylchrysene (5-MeCHR) to study time- and dose-related PAH-DNA binding. The concentrations of 14 PAHs in three extracts were analyzed by HPLC and PAH-DNA adducts were measured by (32)P post-labeling assay. Time-dependent DNA adducts formation of 2.5 microM B[a]P was lower than that of 2.5 microM 5-MeCHR. In comparison with B[a]P, 2-fold higher adduct formation by 5-MeCHR was observed at 12 h exposure, after which BPDE adducts decreased and 5-MeCHR continued to form adducts linearly during 48 h exposure. The data for these two PAH compounds demonstrate a large variation in adduct-forming potency, which should be taken into account when estimating DNA adducts formed by mixtures of unknown PAHs. A clear dose-response effect on formation of DNA adducts was obtained for B[a]P and a Standard Reference Material (SRM) of diesel particulate matter. The amount of B[a]P contributed more to total DNA adduct formation by SRM than by three diesel extracts. Thus, no conclusions can be drawn from diesel particle-derived B[a]P as to the adduct-forming potency of other carcinogenic PAHs. There was little change in adduct levels formed by three diesel extracts from 0 to 12 h exposure. Thereafter, the number of adducts formed by RD2 increased more rapidly than those formed by RD1 and EN97. The concentrations of 14 PAHs and adduct levels analyzed at 24 and 48 h did not change in the same proportion between the extracts. Neither could PAH-DNA adduct levels be explained by the sum of strong and weak adduct-forming PAHs analyzed in the extracts. This indicates that other PAHs in the extracts RD1, RD2 and EN97 contributed to adduct formation more than the carcinogenic adduct-forming PAHs analyzed in this study.     

B[a]P-DNA adduct formation and induction of human epithelial lung cell transformation

In this study we tested the suitability of the human epithelial lung cell line BEAS-2B for in vitro studies of lung carcinogenesis. The human bronchial epithelial lung cell line BEAS-2B, immortalized with an SV-40/Ad-12 hybrid virus construct, was treated for 24 hours with five different concentrations of the lung carcinogen benzo(a)pyrene (B[a]P) to assess the relationship between DNA adduct levels, cell cycle distribution, micronuclei formation (MN), colony forming efficiency (CFE), and anchorage independent growth (AIG). There appeared to be a strong linear correlation between B[a]P concentration and DNA adduct formation, but no difference in cell cycle distribution was observed after incubation with various concentrations of B[a]P. In the incubation range of 4 to 100 nM B[a]P, the number of DNA adducts was linearly correlated with colony formation in AIG and with the number of cells within individual colonies but not the number of colonies in the CFE test. At higher B[a]P concentrations, the clonal expansion of cells in the CFE and the number of colonies in the AIG declined. Also, the number of micronuclei increased with the formation of DNA adducts. It is concluded that after 24 hours of incubation with 100 nM B[a]P, the formation of BPDE-DNA adducts in the human epithelial lung cells BEAS-2B results in maximal induction of cell transformation. Because of this correlation between DNA adduct formation and lung epithelial cell transformation, the BEAS-2B cells seem suitable for in vitro studies on lung carcinogens. Environ. Mol. Mutagen. 30:287–292, 1997 © 1997 Wiley-Liss, Inc.     

Mutability of salmonella tester strains TA1538 (hisD3052) and TA1535 (hisG46) containing the UmuD' and UmuC proteins of escherichia coli

We have studied the mutability of Salmonella typhimurium tester strains carrying plasmids in which either the umuDC or the umuD'C operon of Escherichia coli have been cloned. Reversion of the hisD3052 frameshift mutation by benzo[a]pyrene (B[a]P), aflatoxin B1 (AFB1) and 1-nitropyrene (1-NP), was very efficiently promoted by UmuD' (the activated form of UmuD) and UmuC proteins. In contrast, UmuD'C proteins promoted a moderate reversion of the missense hisG46 allele by B[a]P, and were not effective in mediating this reversion by AFB1. The Salmonella tester strain carrying the hisD3052 allele and containing the E. coli UmuD'C proteins has a sensitivity toward frameshift mutagens similar to that of the MucAB containing strain TA98, and may be useful for obtaining a high level of mutants generated by the SOS mutagenic mechanism in the absence of MucAB proteins. © 1994 Wiley-Liss, Inc.     

Structural features of nitroaromatics that determine mutagenic activity in salmonella typhimurium

Seventeen structurally homologous nitroaromatics were tested for direct-acting mutagenic potency in nine strains of Salmonella typhimurium. The following four structural features were determined to have a strong influence on mutagenic activity: physical dimensions of the aromatic rings, isomeric position of the nitro group, conformation of the nitro group with respect to the plane of the aromatic rings, and ability to resonance-stabilize the ultimate electrophile. Progressive addition of five- and six-membered rings to a nitrobenzene nucleus demonstrated that mutagenic activity was a direct function of size. Fluoranthene was of optimal size (four rings) for mutagenicity; an additional benzene ring, giving benzo[k]fluoranthene, reduced mutagenic activity. Nitroaromatics with a nitro group oriented along the long axis of symmetry of the molecule were more potent mutagens than those with the nitro group oriented along the short axis. These results are discussed in light of the insertion-denaturation model for intercalation of certain DNA adducts. Nitroaromatics with nitro groups sterically forced out of the plane of the aromatic rings were weakly mutagenic or nonmutagenic. Nitro groups located between two peri hydrogens or in a bay-region are examples of this conformation. Finally, structural features that contribute to resonance stabilization of the reactive nitrenium ion enhance mutagenic potency. Thus, 6-nitroindene was at least tenfold more mutagenic than 5-nitroindene. These positional isomers are structurally identical with the exception of the position of an olefinic bond in the adjacent five-membered ring which can contribute to resonance stabilization of a carbonium ion formed after bioactivation of 6-nitroindene but not of 5-nitroindene. The predictive value of these structure-activity relationships should permit a first approximation in the assessment of mutagenic potency of nitroaromatics.     

Identification and in vitro expression of novel CDH23 mutations of patients with Usher syndrome type 1D

Usher syndrome (USH) is a group of autosomal recessive sensory disorders characterized by progressive retinitis pigmentosa (RP) and sensorineural hearing impairment. Usher syndrome type 1 (USH1), with additional vestibular dysfunction, represents the most severe form and shows extensive allelic and non-allelic heterogeneity. At least six USH1 loci exist (USH1A-F), and four of the underlying genes have been identified. Recently, a novel gene, cadherin 23 (CDH23), was shown to be mutated in USH1D. We performed mutation screening by single strand conformation polymorphism (SSCP) analysis and direct sequencing on 33 USH1 patients previously excluded for USH1B and USH1C. On eight disease alleles of four patients, four different mutations were identified, three of them novel (c.6933delT, c.5712G-->A, and IVS45-9G-->A). Exon trapping experiments were performed with two mutations. In the case of a c.5712G-->A transition of the last base of exon 42, that is an apparently synonymous mutation, skipping of exon 42 was observed. By the mutation IVS45-9G-->A, a novel splice acceptor site was created and the insertion of 7 intronic bp was observed. Two mutations, IVS45-9G-->A and the previously described IVS51+5G-->A, were each found in more than one patient. Haplotype analysis by SNPs within CDH23 suggests common ancestors for each of the mutations. Among the total of 52 USH1 cases studied by us, CDH23 mutations account for about 10% of all disease alleles. Our results further suggest that in patients with a typical USH1D phenotype, a significant portion of CDH23 mutations leads to premature termination of translation or loss of numerous amino acid residues, with a high frequency of changes causing aberrant splicing of CDH23 mRNA.     

Beta-thalassemia major caused by compound heterozygosity for +40 to +43(-AAAC), IVS-2-654 (C to T) and codon 41/42 (-TCTT)

OBJECTIVE: To report the analysis of a rare beta-thalassemia ternary heterozygote [+40 to +43(-AAAC)*CD41/42(-TTCT)*IVS-2-654] causing beta-thalassemia major in a Chinese. METHODS: Using PCR-ASO probe hybridization analysis to scan 17 known types of beta-thalassemia mutations, and gene cloning and DNA sequencing to identify the underlying causative mutation. RESULTS: Reverse dot blot (RDB) analysis showed that the patient's beta-globin gene had three mutations: +40 to +43(-AAAC), CD41/42(-TCTT) and IVS-2-654(C to T). Beta-globin gene cloning and sequencing proved that, the two deletions of +40 to +43(-AAAC) and CD41/42(-TCTT) co-existed on the same chromosome, and the other homologous chromosome had an IVS-2-654 (C to T) mutation. So the patient is a compound heterozygote of [+40 to +43(-AAAC)*CD41/42 (-TCTT)]/IVS-2-654 (C to T) leading to beta-thalassemia major. CONCLUSION: The triple mutation of [+40 to +43(-AAAC)*CD41/42(-TCTT)/N] is a new genotype of beta-thalassemia in Chinese.